Antioxidant and cytotoxic potentials of the methanolic extract of Teucrium persicum Boiss. in A-375 melanoma cells

Objective: Teucrium persicum is an Iranian endemic plant used in Iranian traditional medicine. Materials and Methods: The total phenolic and total flavonoid contents, and antioxidant potential of the methanolic extract of T. persicum were determined. The MTT test was used to evaluate the inhibitory effect of the extract on the viability of A-375 cells. The clonogenic, micronucleus formation, and acridine orange/ethidium bromide staining methods were used to evaluate the survival and proliferation of A-375 cells. Apoptosis was evaluated by using DNA fragmentation assay and measuring the activity of caspase 3/7. To study the effect of the extract on the migration of A-375 cells, the in vitro wound-healing (scratch) assay was employed. Results: The average total phenolic and flavonoid contents and antioxidant properties of the extract were 6.97±0.011 mg Ellagic acid (EGA)/g, 46.83±0.0019 mg of the ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline; EQ)/g of dried extract, and 10±0.002 μg/ml, respectively. The IC50 value of the T. persicum methanolic extract was 13 μg/ml for 48 hr. The DNA fragmentation pattern and the activity of caspase3/7 suggested that the reduction of the cell viability may be due to apoptosis induction. Microscopic observations showed nuclear condensation, a considerable increase in micronuclei formation, and inhibition of the colony formation in A-375 cells treated with 7 μg/ml to 15 μg/ml of the extract. Wound-healing assay supported the anti-migration activity of the extract. Conclusion: T. persicum has significant antioxidant and cytotoxic properties. Surely, more detailed molecular and biochemical studies are needed to find the mechanism(s) behind these effects.


Introduction
Melanoma is a malignant skin tumor arising from the malignant transformation of the melanocytes (Das et al., 2016).
Several factors affect susceptibility to melanoma, including genetic and environmental factors (Chinembiri et al., 2014).
The use of plant-derived phytochemicals as therapeutic agents has a long history. Several reports show that some plantderived compounds including vinblastine, vincristine, doxorubicin, topotecan, etc. are used clinically to treat various human cancers (Cragg and Newman, 2005;Hue et al., 2012).
T. persicum grows in Fars province and is used in traditional medicine to alleviate headaches and ventral pains . Several investigations have identified the chemical profile of the essential oils of T. persicum (Javidnia et al., 2007;Miri et al., 2012). A few in vivo studies assessed biological effects of T. persicum (Rasekh et al., 2005;Miri et al., 2015), however, there is no report about the cytotoxic and genotoxic effects of T. persicum on the A-375 cell line as a cancer model. Therefore, in this study, A-375 cells have been used to investigate the cytotoxic and genotoxic potentials of T. persicum.

Methanolic extract preparation
Teucrium persicum was collected from the south of Iran and their identity was confirmed by Dr. Alireza Naqinezhad from the Department of Biology, Faculty of Sciences, University of Mazandaran (9008-HUMZ). Then, 50 g of the aerial parts of T. persicum was powdered and then soaked in 100 ml of pure methanol and shaken for 48 hr. The evaporation process of the supernatant was performed by a rotary evaporator (Heidolph, Germany) at 37ºC. Freeze-drying of the extract was performed by using a freeze-dryer (Christ, Germany). Finally, 50 μg/ml concentration of the extract was prepared as a stock and kept at -20°C for treatments.

Determination of total phenolic contents
The total phenolic contents (TPC) of T. persicum methanolic extract were assessed according to Singleton and Rossi using the Folin-Ciocalteu method (Singleton and Rossi, 1965). Gallic acid was used as a standard, and the TPC was expressed as milligram of gallic acid equivalents/gram of the extract on a dry basis.

Determination of total flavonoid contents
The total flavonoid contents (TFC) of T. persicum methanolic extract were assayed by using the aluminum chloride colorimetric method. Briefly, different concentrations (75-125 µg/ml) of the extract were separately mixed with 1.5 ml of ddH2O and 0.075 ml of 5% NaNO2 for 5 min. Then, 0.15 ml of 10% AlCl3 was added. After 6 min, 0.5 ml of 1M NaOH, and 2.8 ml of distilled water were added and kept at room temperature for 30 min. The absorbance of the mixture was then measured at 510 nm by an ELISA reader (BioTek, USA). Quercetin was used as a standard reference, and the TFC was expressed as milligram of quercetin equivalents per gram of the extract on a dry basis. All assays were accomplished in triplicates.
Evaluating the 2, 2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging potential One milliliter of 0.1 mM solution of DPPH radical in methanol was added to different concentrations (20, 50, and 100 µg/ml) of T. persicum methanolic extract. The mixture was vortexed vigorously and incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm by an ELISA reader (BioTek, USA), and the percentage of scavenging activity (SDPPH) was calculated by the following formula: A blank and A sample indicate the absorbance of the control reaction, and the test compound, respectively. The extract concentration, providing 50% of DPPHradical scavenging activity (IC50), was calculated from a graph, plotted for SDPPH against different concentrations of the extract. All assays were performed in triplicates.

Evaluation of the antioxidant potential of the extract by the Ferric Reducing Antioxidant Power (FRAP) assay
Different concentrations of T. persicum methanolic extract (25, 75, and 125 µg/ml) were added to 2 ml of distilled water and 3 ml of the FRAP reagent, vortexed vigorously, and incubated for 10 min at 37°C. The absorbance of the samples was then measured at 593 nm by an ELISA reader (BioTek, USA). Quercetin and aqueous solution of FeSO4 were used as positive control and standard, respectively. All assays were accomplished in triplicates.

Cell lines and cell culture
A-375 and Human embryonic kidney 293 (HEK-293) cells were purchased from the National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran, Iran). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Sigma) and 1% penicillin/streptomycin (Sigma) and incubated in a 5% CO2 humidified incubator at 37°C. In this study, we used the HEK-293 cells as normal cells to compare the cytotoxicity potential of the extract.

DNA fragmentation assay
In a 6-well plate, A-375 cells were cultured at a density of 6×10 3 cells/well. The cells were then treated with 2, 5, 7, 10, 13, and 15 μg/ml of the extract for 48 hr. After harvesting and collecting cells with 5% Ethylene diamine tetra acetic acid (EDTA)/Trypsin, 400 µl of cell lysis buffer (0.01 M Tris-HCl, pH 8.0, 0.1 M NaCl, 0.025 M EDTA, pH 8, 1% Sodium dodecyl sulfate (SDS) (w/v)) and 0.3 mg/ml of proteinase K were added to pellets and then, incubated overnight at 50°C. Then, 10 µg/ml of RNase was added to the lysate and incubated for 1 hr at 37°C. After centrifugation at 13200 rpm, the supernatant was extracted by using phenol/chloroform/isoamyl alcohol (25:24:1). DNA in the supernatant was precipitated using ethanol for 2 hr at -20 ᵒC and centrifuged at 13200 rpm for 3 min. The dry pellet was dissolved in 15 µl of sterile water, loaded in 1% agarose gel and electrophoresed for one hour.

Acridine orange/ethidium bromide (AO/EtBr) nuclear staining
A-375 cells were seeded on gelatincoated coverslips in 6-well plates for 48 hr. After that, cells were treated with 5, 10, 13, and 15 µg/ml of the extract for 48 hr. The cells were rinsed with PBS and fixed with 50:50 v/v of cold acetone-methanol solution for 15 min at -20°C, and then, rinsed with PBS twice. A solution containing 100 µg/ml of AO and 100 µg/ml of EtBr (Sigma) was added. After 30 min of incubation, the coverslips were washed with PBS and air-dried and then viewed under a fluorescence microscope (Nikon, Japan).

Clonogenic assay
A-375 cells were seeded at a density of 2×10 2 cells/well in a 24-well plate for 48 hr to attach to the dishes. The cells were treated with 5, 10, 13, 15, and 20 µg/ml of the extract for 7 days. The colonies were fixed with cold methanol-acetic acid (7:1) for 15 min and then, stained with crystal violet (0.5%) for 2 hr. Afterward, the stain was removed, and samples were rinsed with tap water, and allowed to air-dry. Finally, colonies were checked by a stereomicroscope (Olympus, Japan). All assays were performed in triplicates.

Micronucleus assay
In a 24-well plate, A-375 cells were seeded at a density of 1.2×10 4 cells/well for 48 hr, then, treated with 2, 5, 7, 10, 13, 15, and 20 µg/ml of the extract. After 15 hr, 4 µg/ml of cytochalasin B was added to each well for 28 hr. The cells were then placed in a cold hypotonic solution containing 0.57% KCl and fixed with cold methanol-glacial acetic acid (6:1) mixture. After 48 hr, cells were stained with a solution of 15 % Giemsa (Merck, Germany) in a PBS buffer (pH 7.4) for 15 min and allowed to air-dry. The cells were then viewed and scored by light microscopy at 400× magnification. All assays were performed in triplicates.

In vitro wound-healing assay
A-375 cells were plated at a density of 6×10 3 cells/well in a 6-well plate and grown to confluence. Next, by using a sterile plastic yellow tip, a scratch was created in the monolayer. To remove damaged or floating cells, the medium was then removed, and the cells were washed with PBS. The cells were treated with 2, 4, 6, 8, and 10 µg/ml of the extract for 48 hr. The plates were then examined by light microscopy for their migration ability. All assays were performed in triplicates.

Detection of caspases 3/7 activity
The caspase-3/7 activity was determined using a caspase assay kit according to the instructions of the manufacturer (Kiazist, Iran). A-375 cells were seeded at a density of 4×10 4 cells/well in a 6-well plate, grown to 70% confluency, and treated with 7, 10, 13, 15, and 17 µg/ml of the methanolic extract of T. persicum for 48 hr. The cells were harvested and centrifuged at 1500 rpm for 3 min. The supernatant was discarded, cell lysis buffer, Dithiothreitol (DTT), caspase buffer, and caspase substrate were added to each well and incubated for 2 hr at 37ᵒC. Finally, the light absorbency was measured at 405 nm by an ELISA reader. All assays were performed in triplicates.

Statistical analyzes
The data are presented as means ± standard deviations from the three replicates. Statistical analyses were conducted using SPSS (IBM Statistics 25.0). A p-value <0.05 was considered statistically significant. The IC50 value was calculated by Excel (Office 2016) and GraphPad Prism software version 8 (California, USA). The area of wound surfaces was measured by using ImageJ software ver. 1.40g (NIH, Bethesda, MD, USA).

Radical scavenging activity of T. persicum methanolic extract
To evaluate the free radical scavenging activity of T. persicum, DPPH was used as a stable free radical and ascorbic acid as a standard. In the DPPH free radical scavenging assay, 50% inhibition was observed at a concentration of 10±0.002 μg/ml of T. persicum methanolic extract, while the IC50 value of ascorbic acid was 5±0.005 μg/ml (p<0.05).

Antioxidant potential of T. persicum methanolic extract
The antioxidant potential of T. persicum extract was evaluated using the FRAP assay. The average ferric reducing antioxidant power for T. persicum methanolic extract and quercetin was 100.25±0.003 and 81.08±0.01 µM of Fe 2+ , respectively (p<0.05).

Effect of T. persicum extract on the viability of A-375 cells
A-375 cells were treated with 5, 10, 25, 50, 75, 100, 125, 150, 175, and 200 μg/ml of the extract. The IC50 value of T. persicum extract for A-375 cells was 13 μg/ml for 48 hr (Figure 1). Treatment of A-375 cells with 5 μg/ml of the extract led to an insignificant increase in the population of viable cells (less than 10%), but higher concentrations reduced the cell viability in a dosedependent manner (p<0.01) ( Figure 1A). Due to the lack of access to the normal human skin cell line, we used the HEK-293 cell line as a replacement. The IC50 value of the extract for HEK-293 cells was 49.94 μg/ml (p<0.01) ( Figure 1B).

Effect of T. persicum extract on the genomic DNA of A-375 cells
A-375 cells were treated with 2, 5, 7, 10, 13, and 15 μg/ml of the extract for 48 hr, and then, genomic DNA was extracted. Electrophoresis of genomic DNA extracted from each sample in a 1% agarose gel was performed and it was followed by staining with the ethidium bromide. A typical DNA ladder was never observed, while DNA degradation always appeared as a smear in the gel. The smear disappeared in cells treated with higher concentrations including 10, 13, and 15 µg/ml, due to a considerable decrease in the number of viable cells (Figure 2).

Effect of methanolic extract of T. persicum on the nuclear status of A-375 cells
A-375 cells that were treated with sublethal or lethal concentrations of the extract were stained with Acridine orange/Ethidium bromide solution. No significant nuclear condensation was detected in control cells, but the cells incubated with concentrations near to or above the IC50 (10, 13, and 15 µg/ml of the methanolic extract) for 48 hr showed nuclear condensation (Figure 3).   Figure 4 exhibits the clonogenic survival of A-375 cells treated with 5, 10, 13, 15 µg/ml of the methanolic extract. A reduction in cell division was observed in cells treated with 5 µg/ml of the extract, and 10, 13, and 15 µg/ml of the extract led to suppression of the colony-forming capacity in A-375 treated cells. The plating efficiency (PE) was 57.76% for the control group (non-treated cells), and a surviving fraction (SF) of 1 was calculated for 5 µg/ml of the extract (p<0.01) (Figure 4).

The extract induced micronucleus formation in A-375 cells
It is interesting to know that the extract showed genotoxic effects on the cytokinesis of A-375 cells. Our microscopic observations demonstrated that A-375 cells treated with a concentration of 2 µg/ml of the extract were not much different from the control group, but the frequency of binucleated and multinucleated cells or micronuclei in cells treated with 5, 7, 10, 13, and 15 µg/ml of the extract was significantly more than the controls (nontreated) cells ( Figure 5).

T. persicum extract inhibited the invasion of A-375 cells
To find out whether T. persicum extract had any effect on the invasion of A-375 cells, we used the wound-healing (scratch) assay. Figure 6A presents that T. persicum extract reduced the proliferation and invasion of A-375 cells in a dose-dependent manner. The wound closure values in cells treated with 0 (non-treated) 2, 4, 6, 8, and 10 μg/ml of the extract were 100, 59, 47.1, 35.4, 17.5, and 13.9%, respectively (p<0.05) ( Figure 6B).

T. persicum extract induced the activity of caspases 3/7 in A-375 cells
To examine the activity of caspases 3 and 7 levels, A-375 cells were treated with lethal doses of T. persicum methanolic extract for 48 hr. As shown in Figure 7, treatment of A-375 cells with concentrations of 13, 15, and 17 μg/ml of the extract in comparison with untreated cells significantly induced the activity of caspases 3 and 7 in a concentrationdependent manner (p<0.05).

Discussion
In this study, we aimed to investigate the antioxidant and cytotoxic properties of T. persicum, as an Iranian endemic species.
Our biochemical analyses showed that T. persicum methanolic extract contains significant total phenolic and flavonoid contents that are responsible for its remarkable antioxidant and radical scavenging properties. We found that 10±0.002 μg/ml of the methanolic extract is required to scavenge 50% of DPPH radicals (compared to 5±0.005 μg/ml of ascorbic acid) (p<0.05). A previous report indicated that Teucrium species are rich in phenolic and flavonoid compounds with very strong biological activities (Stankovic et al., 2011). In a comparative study, it was found that methanolic extract of T. persicum has higher phenolic and flavonoid contents and antioxidant activity than chloroform, ethyl acetate, and water extracts (Miri et al., 2012). In another study, the essential oil of T. persicum showed significant free radical scavenging and antioxidant activities .
The results of DNA fragmentation assays revealed that the reduction in the viability of A-375 cells is due to apoptosis induction. DNA fragmentation started at the concentration of 5 µg/ml of the extract and continued at higher concentrations. Interestingly, at the concentration of 2 µg/ml of the extract where a slight increase in the cell viability occurred, DNA fragmentation was not observed (Figure 2). Caspases 3 and 7 are both activated during apoptosis, irrespective of the deathinitiating stimulus (Walsh et al., 2008). The caspase activating potential of T. persicum extract has not been investigated, yet. In this study, we found that treatment of A-375 cells with T. persicum methanolic extract led to the induction of caspase 3/7 activity in a dose-dependent manner (starting at 7 µg/ml, p<0.05) (Figure 7). Several studies have shown that plant extracts or plant-derived compounds possess apoptosis induction potential via induction of caspase 3/7, 8, and 9 activities (Das et al., 2012;Safarzadeh et al., 2014;Panicker et al., 2019). AO/EtBr staining results showed that treatment of A-375 cells with concentrations higher than IC50 resulted in nuclear condensation that is one of the hallmarks of apoptosis (Figure 3).
The results of the clonogenic assay showed that T. persicum extract significantly reduced proliferation and colony formation capacity of A-375 cells in a concentration-dependent manner (p<0.01) (Figure 4). In a similar study, it has been shown that T. polium extract decreased colony formation potential in some cancer cell lines (Nematollahi-Mahani et al., 2007;Rajabalian, 2008). Caryophyllene oxide, linalool, α-pinene, αcadinene, 1,4-cadinadiene, and α-terpinyl acetate are the main chemical components of T. persicum essential oil (Javidnia et al., 2007;Miri et al., 2012). According to reports published, α-pinene, βcaryophyllene, and β-caryophyllene-oxide have cytotoxic effects against various cancer cells (Dahham et al., 2015;Salehi et al., 2019). Laboratory investigations suggest that there are one or more compounds in essential oils of Teucrium species that may work synergistically to inhibit cancer cells (Stankovic et al., 2011;Al-Hamwi et al., 2021). The micronucleus technique is a well-known and sensitive method to assess the genotoxic effects and chromosomal damages potentials of various compounds (Fenech, 2000). Although evidence shows genotoxicity potentials of other Teucrium species (Milošević-Djordjević et al., 2013;Grujičić et al., 2020), the genotoxic potentials of T. persicum have not been investigated yet. The results of our study showed that T. persicum extract has significant genotoxic potential and induced micronucleus formation in A-375 cells ( Figure 5). Therefore, it is inferred that the inhibitory effect of T. persicum extract on the proliferation machinery of A-375 cells may be due to its genotoxic effects.
Cell proliferation and invasion have a major impact on the wound-healing process (Liang et al., 2007). However, the sublethal concentrations (2 and 4 µg/ml) of the extract did not show a remarkable effect on the cell viability, but treatment of A-375 cells with the extract led to a notable decrease in the wound-healing process in the cell sheets (p<0.05) (Figure 6). Analyzing the phytochemical profile of essential oils prepared from Teucrium species revealed that the presence of polyphenols including gallic acid, caffeic acid, quercetin, kaempferol, luteolin, apigenin, etc. may speed up the woundhealing process (Meguellati et al., 2019;Fallah Huseini et al., 2020;Chabane et al., 2021). Therefore, further investigations are needed to prove it and then conclude that probably there are one or more compounds in T. persicum that can inhibit the invasion of A-375 cells.
In conclusion, this is a preliminary investigation on the genotoxic and cytotoxic properties of T. persicum methanolic extract on A-375 cancer cells. The results of this study indicated that T. persicum has significant antioxidant and cytotoxic effects and can be a good candidate for future investigations. Further analyses on the chemical composition of T. persicum extract boost certainly the process of studying its anti-cancer potentials as well as investigating the underlying molecular mechanism(s).